We are searching for the mechanism that makes cells more vulnerable to environmental exposures when the cells are located at the earliest part of the S phase, when DNA replication is being initiated. We have observed that replicating DNA is more vulnerable to attack by chemical carcinogens than the bulk of the nuclear DNA. Furthermore, DNA damage occurring in the early part of the S phase is far more effective than damage at other times in producing transformation of cells. Therefore, we believe that DNA sites replicating during the early S phase are probably the sites in DNA most damaged by treatments at this time. From this we presumed that this early replicating DNA fraction included DNA segments that are essential to the transformation process. The vastly higher transformation rates result from the greater likelihood that these DNA sites would be damaged and genetically altered as the result of treatments with carcinogens early in the S phase. The goal of this project is to identify sites in DNA that might be critical to transformation. The concept is that DNA replicated early in the S phase should include DNA segments close to and at replication origins. Alteration of such sites in DNA could offer means whereby the fine regulation of genomic DNA replication could be degraded. Based on observations that DNA replication occurs and starts at sites physically organized and related to the nuclear matrix, we propose a search strategy for such START sites based on their presence in both early S DNA libraries and matrix-associated DNA (matrix associated in late G1 or G1/S transition border) libraries. Studies are proposed to evaluate the selected clones based on their ability to bind to DNA-free nuclear matrix and their ability to bind replication proteins selectively based on their recovery from the G1/S border of the cycle versus early G1/G0. We will also evaluate whether the clones can initiate and sustain DNA replication in vitro with nuclear protein extracts or whether the human DNA insert could act as an origin of replication for the plasmids transfected into human cells. In addition, studies will be undertaken to assess whether genomic DNA corresponding to these sites are altered in transformed cells, or even early stage, partially transformed cells as compared to normal cells.